Structures of the ON and OFF states of the membraneous fumarate/Citrate sensor kinases DcuS from E. coli and CitA from Geobacillus thermodenitrifcans

传感器激酶（SK）是细菌响应细胞外刺激的主要装置，导致SK的自磷酸化。它们的功能包括信号感知，跨膜（TM）信号传导和信号传递到激酶。SK的结构域组成和跨膜信号传导表现出广泛的变异。该提案旨在解决DcuS/CitA家族（富马酸盐和柠檬酸盐传感）的SK在ON和OFF状态下的结构，并了解跨膜和细胞内信号传导中的结构域的重组。DcuS/CitA包含跨膜信号SK的典型结构域组成。从大量的TM传感器激酶中，NarQ硝酸盐SK的结构最近已经以一种耗尽细胞质GAF和激酶结构域的形式被解决。NarQ代表一种独特的SK，其结构域组成与dcu /CitA有很大不同，很可能代表一种不同的信号转导模式。大肠杆菌的dcu和与之密切相关的嗜热反硝化地杆菌的CitA都是同二聚体，由一个用于富马酸盐或柠檬酸盐结合的胞外质PASP （Per ARNT Sim）结构域、两个TM螺旋（TM1、TM2）、一个胞质PASC和激酶结构域组成。根据分析系统中蛋白质的可用性，用dcu或CitA进行互补研究。PASP和PASC的结构已经在开和关状态下以孤立的和膜积分的结构求解。结构、遗传和生化研究表明，底物结合时PASP的压实会触发TM2的活塞状运动，TM2负责跨膜信号传导。PASC在α -螺旋二聚化位点的结构重排中响应TM2的移位。这项工作是高分辨率分析和跨膜信号传导过程中结构重排的综合观点的基础。工作将集中在(i) TM2的活塞式运动可能与TM2的剪刀式运动相结合的TM区域的结构重排，（ii） PASc在信号传递和激酶结构域控制过程中的结构重排。为了获得完整的视图和高分辨率，将采用结构（NMR和结晶；AG Griesinger），生化，遗传（AG Unden）和生物物理（AG Hinderberger, Griesinger）方法。准备工作建立了研究的所有结构和方法，我们认为DcuS/CitA代表了极少数通过结构生物学、生物化学和生物物理学的结合方法可以达到高结构和功能分辨率的跨膜和细胞质信号传导的SK之一。

Sensor kinases (SK) represent a major device of bacteria for responding to extracellular stimuli, resulting in autophosphorylation of the SK. Their function includes signal perception, transmembrane (TM) signaling, and signal transmission to the kinase. Domain composition and transmembrane signaling of the SK show broad variation. The proposal aims at solving the structure of a SK of the DcuS/CitA family (fumarate and citrate sensing) in the ON and the OFF state, and to understand restructuring of the domains in transmembrane and intracellular signaling. DcuS/CitA contain an archaetypical domain composition for transmembrane signaling SK. From the vast number of TM sensor kinases, the structure of the NarQ nitrate SK has been solved in a form depleted of the cytosolic GAF and kinase domains recently. NarQ represents a unique SK with a domain composition largely different from DcuS/CitA and stands very likely for a different mode of signal transduction. DcuS of E. coli and the closely related CitA of the thermophilic Geobacillus thermodenitrificans are homodimers each, composed of an extracytoplasmic PASP (Per ARNT Sim) domain for fumarate or citrate binding, two TM helices (TM1, TM2), a cytoplasmic PASC and the kinase domain. Studies are performed with DcuS or CitA complementarily according to availability of the proteins for the analytic systems. Structures of PASP and PASC have been solved in isolated and membrane-integral constructs in the ON and OFF states. Structural, genetic and biochemical studies show a compaction of PASP upon substrate binding that triggers a piston-like movement of TM2 which is responsible for transmembrane signaling. PASC responds to the shift of TM2 in a structural rearrangement at an alpha-helical dimerization site. This work is the basis for high resolution analysis and an integrated view of the structural rearrangements in the domains during transmembrane signaling. Work will concentrate on (i) on the structural rearrangements in the TM region with the piston-like movement of TM2 that may be combined with a scissors movement of TM2, (ii) the structural rearrangement in PASc during signal transmission and control of the kinase domain. For a complete view and high resolution, structural (NMR and crystallization; AG Griesinger), biochemical, genetic (AG Unden) and biophysical (AG Hinderberger, Griesinger) methods will be applied. Preparatory work established all constructs and the methods for the study, and we presume that DcuS/CitA represent one of the very few SK whose transmembrane and cytoplasmic signaling is within reach at high structural and functional resolution by a combined approach from structural biology, biochemistry and biophysics.